This invention relates to human thrombomodulin and, more particularly, to the cDNA clone representing the full size human thrombomodulin.
Thrombomodulin is an endothelial cell surface thrombin-binding glycoprotein which converts thrombin into a protein C activator. Activated protein C then functions as an anticoagulant by inactivating two regulatory proteins of the clotting system, namely factors Va and VIIIa. The latter two proteins are essential for the function of two of the coagulation proteases, namely factors IXa and Xa. Thrombomodulin thus plays an active role in blood clot formation in vivo and can function as a direct or indirect anticoagulant.
Thrombomodulin has been purified from rabbit [Esmon et al., J. Biol. Chem. 257, 859-864 (1982)], bovine [Suzuki et al., Biochim. Biophys. Acta 882, 343-352 (1986); Jakubowski et al., J. Biol. Chem. 261, 3876-3882 (1986)], human lung [Maruyama et al., J. Clin. Invest. 75, 987-991 (1985)] and human placenta [Salem et al., J. Biol. Chem. 259, 12246-12251 (1984)]. The human protein has an apparent M.sub.r =75,000 (unreduced) that exhibits a characteristic shift to M.sub.r =100,000 upon reduction with 2-mercaptoethanol. Immunohistochemical examination of tissue sections revealed that thrombomodulin is widely distributed in the endothelium of arteries, veins, capillaries, and lymphatics [Maruyama et al., J. Cell Biol. 101, 363-371 (1985)].
Recent advances in biochemistry and in recombinant DNA technology have made it possible to synthesize specific proteins, for example, enzymes, under controlled conditions independent of the organism from which they are normally isolated. These biochemical synthetic methods employ enzymes and subcellular components of the protein synthesizing systems of living cells, either in vitro in cell-free systems, or in vivo in microorganisms. In either case, the principal element is provision of a deoxyribonucleic acid (DNA) of specific sequence which contains the information required to specify the desired amino acid sequence. Such a specific DNA sequence is termed a gene. The coding relationships whereby a deoxyribonucleotide sequence is used to specify the amino acid sequence of a protein is well-known and operates according to a fundamental set of principles. See, for example, Watson, Molecular Biology of the Gene, 3d ed., Benjamin-Cummings, Menlo Park, Calif., 1976.
A cloned gene may be used to specify the amino acid sequence of proteins synthesized by in vitro systems. RNA-directed protein synthesizing systems are well-established in the art. Double-stranded DNA can be induced to generate messenger RNA (mRNA) in vitro with subsequent high fidelity translation of the RNA sequence into protein.
It is now possible to isolate specific genes or portions thereof from higher organisms, such as man and animals, and to transfer the genes or fragments to microorganisms such as bacteria or yeasts. The transferred gene is replicated and propogated as the transformed microorganism replicates. Consequently, the transformed microorganism is endowed with the capacity to make the desired protein or gene which it encodes, for example, an enzyme, and then passes on this capability to its progeny. See, for example, Cohen and Boyer, U.S. Pat. Nos. 4,237,224 and 4,468,464.